Transformed plant and flowering regulation method using flowering-inducing gene

ABSTRACT

Novel sugarcane-derived flowering-inducing genes each encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 (the ScFT6 gene and the ScZCN16 gene), by which the flowering time is accelerated more slowly than conventionally known flowering-inducing genes, are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from Japanese patent applicationJP 2019-039843 filed on Mar. 5, 2019, the content of which is herebyincorporated by reference into this application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 12, 2020 isnamed Sequence Listing_ST25 94369-13_ST25.txt and is 23 KB is size.

BACKGROUND Technical Field

The present disclosure relates to a transformed plant that has acquiredimproved properties by introducing a certain flowering-inducing genethere into and a flowering regulation method using theflowering-inducing gene.

Background Art

Conventionally, cross breeding of plants has been carried out with acombination based on experience and intuition, and a large number ofprogeny lines has been selected by comprehensive evaluation. Usually, inorder to carry out crossing, it is necessary to go through a process offlowering induction, blooming/pollination, and seed settingpromotion/seed harvesting. Depending on the plant type, this process canbe scheduled only once a year, even in an area suitable for cultivation.Therefore, it took a very long time to develop one cultivar.Furthermore, when the cultivar to be bred was a cultivar that isdifficult to flower or when the cultivars to be bred did not match interms of time of blooming, it was very difficult to carry out desiredcrossing.

As explained above, in cross breeding, it was desired to develop atechnology that would speed up the time of blooming by controlling theblooming of plants. It has been reported that it was possible to induceflowering (heading) of Arabidopsis thaliana or rice by causing aflowering-inducing gene such as the FT gene (AtFT gene) or the Hd3a gene(OsHd3a gene) to be overexpressed (JP 2000-139250 A, JP 2002-511270 A,and JP 2002-153283 A; Kardailsky I. et al., Science. 1999 Dec. 3;286(5446):1962-5 and Kojima S. et al., Plant Cell Physiol 2002 October;43(10):1096-105).

Meanwhile, in the case of using these flowering-inducing genes, as thegenes have very powerful ability to induce flowering, flowering occursat a stage when a plant is still small, and thus, the amount of seedsthat can be harvested decreases, which has been problematic. JP2008-525013 A and JP 2002-537768 A disclose a technology that controlsthe flowering time using a gene encoding a regulatory factor forcontrolling the expression of the flowering-inducing gene. However, asthe technology disclosed in JP 2008-525013 A and JP 2002-537768 A causesthe introduced regulatory factor to affect genes other than theflowering-inducing gene, undesirable characteristics may be imparted.

In addition, JP Patent No. 5828302 discloses a technology related tosugarcane into which the rice flowering-inducing gene Hd3a has beenintroduced to change the flowering time. However, even the technologydisclosed in JP Patent No. 5828302 is also problematic because as thegene has very powerful ability to induce flowering, flowering occurs ata stage when a plant is still small.

Further, US2018/0057831 A1 discloses a technology that regulates theflowering time by allowing the expression of a flowering-inducing genedownstream of an alcohol-induced promoter. However, according to thetechnology disclosed in US2018/0057831 A1, the promoter needs to beactivated with the aid of alcohol, which causes a problem that floweringregulation is time- and cost-consuming. Furthermore, WO2011/115222 A1discloses a technology that regulates the flowering time by introducinga mutation into a certain site of the protein encoded by the Hd3a geneof rice. However, according to the technology disclosed in WO2011/115222A1, there is a problem that production of such mutation gene is time-and cost-consuming. Moreover, US2011/0257013 A1 discloses a technologythat regulates the flowering time by allowing the expression of aflowering-inducing gene downstream of a copper ion-inducible promoter.However, according to the technology disclosed in US2011/0257013 A1, thepromoter needs to be activated with the aid of the copper ion-induciblepromoter, which causes a problem that flowering regulation is time- andcost-consuming.

SUMMARY

As described above, flowering of a plant cannot be regulated at thedesired time even by simply introducing a flowering-inducing gene intothe plant. The flowering time can be regulated only by a method using aninducible promoter for regulating the expression of a flowering-inducinggene or utilizing a mutant-type flowering-inducing gene, which has beenproblematic.

In consideration of the above-described circumstances, the presentdisclosure provides a transformed plant having a novelflowering-inducing gene introduced thereinto, the gene having ability toinduce flowering so as to function for accelerating the flowering timemore slowly than conventionally known flowering-inducing genes, and aflowering regulation method using the flowering-inducing gene.

For example, introducing a certain flowering-inducing gene makes itpossible to induce flowering more slowly than when a conventionallyknown flowering-inducing gene is introduced.

(1) A transformed plant or transformed plant cell, wherein aflowering-inducing gene encoding any one of the following proteins (a)to (c) has been introduced thereinto or wherein an expression of theflowering-inducing gene present as an endogenous gene has been enhanced.

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4;

(b) a protein comprising an amino acid sequence having 90% or moreidentity to the amino acid sequence of SEQ ID NO: 2 or 4 and havingability to induce flowering; and

(c) a protein comprising an amino acid sequence encoded by apolynucleotide capable of hybridizing with all or part of apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3under stringent conditions and having ability to induce flowering.

(2) The transformed plant or transformed plant cell according to (1),which belongs to the family Poaceae.

(3) The transformed plant or transformed plant cell according to (1),which belongs to the genus Saccharum, Erianthus, Sorghum, or Miscanthus.

(4) A flowering induction method, comprising introducing aflowering-inducing gene encoding any one of the following proteins (a)to (c) or enhancing the expression of the flowering-inducing genepresent as an endogenous gene:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4;

(b) a protein comprising an amino acid sequence having 90% or moreidentity to the amino acid sequence of SEQ ID NO: 2 or 4 and havingability to induce flowering; and

(c) a protein comprising an amino acid sequence encoded by apolynucleotide capable of hybridizing with all or part of apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3under stringent conditions and having ability to induce flowering.

(5) The flowering induction method according to (4), which comprisesintroducing the flowering-inducing gene into a plant belonging to thefamily Poaceae.

(6) The flowering induction method according to (4), which comprisesintroducing the flowering-inducing gene into a plant belonging to thegenus Saccharum, Erianthus, Sorghum, or Miscanthus.

According to the present disclosure, using a novel flowering-inducinggene makes it possible to promote flowering more slowly than when aconventionally known flowering-inducing gene is introduced. Therefore,in the case of the transformed plant or transformed plant cell accordingto the present disclosure, flowering induction occurs at a stage when aplant has grown more largely than when a conventionally knownflowering-inducing gene is introduced. Accordingly, the transformedplant or transformed plant cell according to the present disclosure hasan improved feature that the flowering time is accelerated whileensuring a sufficient seed yield.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic diagram showing the results of investigatingflower bud formation when transformed plants prepared by separatelyintroducing FT family genes into the FT function-deficient strain werecultivated under long-day conditions;

FIG. 2 is a characteristic diagram showing the results of investigatingseed yield when transformed plants prepared by separately introducing FTfamily genes into the FT function-deficient strain were cultivated underlong-day conditions;

FIG. 3 is a characteristic diagram showing the results of investigatingflower bud formation (based on the number of leaves upon flower budformation) when transformed plants prepared by separately introducing FTfamily genes into the wild-type strain were cultivated under short-dayconditions; and

FIG. 4 is a characteristic diagram showing the results of investigatingflower bud formation (based on the number of days until flower budformation) when transformed plants prepared by separately introducing FTfamily genes into the wild-type strain were cultivated under short-dayconditions.

DETAILED DESCRIPTION

The present disclosure will be described in detail below.

According to the present disclosure, a certain flowering-inducing geneis introduced into a plant cell to serve as a host or an expression ofthe flowering-inducing gene present as an endogenous gene is enhanced.Accordingly, flowering induction occurs in a transformed plant havingthe flowering-inducing gene introduced thereinto or showing enhancedexpression of the flowering-inducing gene earlier than floweringinduction in a wild-type plant not introducing the flowering-inducinggene or not showing the enhanced expression of the gene. Meanwhile,flowering induction occurs in the transformed plant later than floweringinduction in a transformed plant having a conventionally knownflowering-inducing gene introduced thereinto or showing enhancedexpression of the conventionally known flowering-inducing gene presentas an endogenous gene. In the following explanation, a phenomenon inwhich flowering induction occurs earlier than flowering induction in awild-type plant, but later than flowering induction in a transformedplant having a conventionally known flowering-inducing gene introducedthereinto or showing enhanced expression of the conventionally knownflowering-inducing gene present as an endogenous gene is expressed as,for example, “slow flowering induction” or “flowering induction occursslowly.”

The term “flowering induction” used herein refers to transition from thevegetative growth phase to the reproductive growth phase, which meansformation, differentiation, and development of flower buds that occurbefore blooming. In addition, flowering is induced as a result ofincreased expression of various related genes caused by the formation ofa complex of a florigen transported to the shoot apex through thevascular phloem. Therefore, the flowering induction time can also bejudged by observing the formation, differentiation, and development offlower buds, by detecting the presence of a florigen or a florigenactivation complex, or by detecting the transcript of a gene which isincreasingly expressed by the florigen activation complex.

Flowering-Inducing Genes Involved in Slow Flowering Induction

The flowering-inducing gene involved in slow flowering inductionaccording to the present disclosure is a gene encoding a proteincomprising the amino acid sequence of SEQ ID NO: 2 or 4. Genes eachencoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or4 are genes that have been newly identified from sugarcane based onsequence information on conventionally known flowering-inducing genes(the Arabidopsis thaliana-derived FT gene (AtFT gene), the rice-derivedflorigen gene (Hd3a gene), and the corn-derived florigen gene (ZCN8gene)) explained in the Examples described later. Note that theflowering-inducing gene has, as a coding region, the nucleotide sequenceof SEQ ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 2 or thenucleotide sequence of SEQ ID NO: 3 encoding the amino acid sequence ofSEQ ID NO: 4.

In the Examples described later, the flowering-inducing gene identifiedwith the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequenceof SEQ ID NO: 2 is referred to as “ScFT6 gene” and theflowering-inducing gene identified with the nucleotide sequence of SEQID NO: 3 and the amino acid sequence of SEQ ID NO: 4 is referred to as“ScZCN16 gene.”

In addition, examples of the flowering-inducing gene involved in slowflowering induction according to the present disclosure include geneshomologous to the gene encoding the protein of SEQ ID NO: 2 or 4. Thesehomologous genes include both genes that have evolved and branched froma common ancestor gene and genes that simply have similar nucleotidesequences, unlike the evolved and branched genes. Genes that haveevolved and branched from a common ancestor gene include homologousgenes (orthologs) of two different species and homologous genes(paralogs) that are generated within one species due to duplication.Genes homologous to the above-described flowering-inducing gene can bereadily searched for or identified based on the amino acid sequence ofSEQ ID NO: 2 or 4 using a known database such as GenBank.

Further, the flowering-inducing gene involved in slow floweringinduction according to the present disclosure is not limited to a geneencoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or4 and may be a gene encoding a protein comprising an amino acid sequencehaving 80% or more identity, 85% or more identity in some embodiments,90% or more identity in some other embodiments, 95% or more identity instill some other embodiments, and 98% or more identity in yet some otherembodiments to the amino acid sequence of SEQ ID NO: 2 or 4. Note that aprotein having an amino acid sequence that is different from the aminoacid sequence of SEQ ID NO: 2 or 4 functions to cause slow floweringinduction when it is expressed in a host plant. Here, the value ofidentity is a value obtained with default settings using a computerprogram implemented with the BLAST (Basic Local Alignment Search Tool)program and a database storing gene sequence information

Furthermore, the flowering-inducing gene involved in slow floweringinduction according to the present disclosure is not limited to a geneencoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or4 and may be a gene encoding a protein comprising an amino acid sequencein which one or more amino acids are deleted, substituted, added, orinserted in the amino acid sequence of SEQ ID NO: 2 or 4 and functioningto cause slow flowering induction. Here, the expression “one or moreamino acids” means, for example, 1 to 20 amino acids, 1 to 10 aminoacids in some embodiments, 1 to 7 amino acids in some other embodiments,1 to 5 amino acids in still some other embodiments, and 1 to 3 aminoacids in some particular embodiments. Amino acids can be deleted,substituted, or added by modifying a nucleotide sequence encoding aprotein comprising the amino acid sequence of SEQ ID NO: 2 or 4 by atechnique known in the art. A mutation can be introduced into anucleotide sequence by a known method such as the Kunkel method or theGapped duplex method or a method similar to such method. A mutation isintroduced using, for example, a mutagenesis kit (e.g., Mutant-K orMutant-G (trade name, TAKARA Bio Inc.)) or an LA PCR in vitroMutagenesis series kit (trade name, TAKARA Bio Inc.) by thesite-directed mutagenesis method. Mutagenesis may be carried out by amethod using a chemical mutation agent such as EMS (ethylmethanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine, and a different carcinogeniccompound or a method involving radiation treatment or ultraviolettreatment typically with X-ray, alpha ray, beta ray, gamma ray, or ionbeam.

Moreover, the flowering-inducing gene involved in slow floweringinduction according to the present disclosure may be a gene encoding aprotein hybridizing with all or part of a complementary strand of DNAcomprising the nucleotide sequence of SEQ ID NO: 1 or 3 under stringentconditions and functioning to cause slow flowering induction. The term“stringent conditions” used herein refers to conditions under whichso-called specific hybrids are formed while non-specific hybrids are notformed. For example, such conditions include hybridization at 45° C.with 6×SSC (sodium chloride/sodium citrate) and subsequent washing at50° C. to 65° C. with 0.2 to 1×SSC and 0.1% SDS or hybridization at 65°C. to 70° C. with 1×SSC and subsequent washing at 65° C. to 70° C. with0.3×SSC. Hybridization can be carried out by a conventionally knownmethod such as the method described in J. Sambrook et al. MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989).

Expression Vector

An expression vector is constructed such that it includes a nucleic acidhaving a promoter nucleotide sequence that enables constitutiveexpression and the above-described flowering-inducing gene. It ispossible to produce a transformed plant having the flowering-inducinggene introduced thereinto with the use of the expression vector. Inaddition, an expression vector may be one that substitutes an endogenouspromoter for regulating the expression of the flowering-inducing genewith a powerful promoter by homologous recombination in order to enhancethe expression of the flowering-inducing gene present as an endogenousgene. In this case, an expression vector is constructed such that it hasa powerful promoter described in detail later and a region necessary forhomologous recombination.

Various conventionally known vectors can be used herein as a base vectorfor the expression vector. For example, a plasmid, phage, cosmid, or thelike can be used, and a vector can be appropriately selected accordingto a plant cell into which the vector is introduced and a method forintroducing the vector. Specific examples of such vector include pBIvectors such as pBR322, pBR325, pUC19, pUC119, pBluescript, andpBluescriptSK. In particular, in a case in which the method forintroducing the vector into a plant cell uses Agrobacterium, a pBIbinary vector is used in some embodiments. Specific examples of a pBIbinary vector include pBIG, pBIN19, pBI101, pBI121, and pBI221.

The promoter is not particularly limited as long as it is a promoterthat allows the flowering-inducing gene to be expressed in a plant, anda known promoter can be used as appropriate. Examples of such promoterinclude a cauliflower mosaic virus 35S promoter (CaMV35S), various actingene promoters, various ubiquitin gene promoters, a nopaline synthasegene promoter, a tobacco PR1a gene promoter, a tomato ribulose1,5-bisphosphate carboxylase/oxidase small subunit gene promoter, anapin gene promoter, and an oleosin gene promoter. Of these, acauliflower mosaic virus 35S promoter, an actin gene promoter, or anubiquitin gene promoter is used in some embodiments. The use of each ofthe above-described promoters allows an arbitrary gene to be stronglyexpressed when the gene is introduced into a plant cell.

In addition, a promoter that functions to allow a nucleic acid to beexpressed in a plant in a site-specific manner can also be used. Anyconventionally known promoter can be used as such promoter. Byintroducing the flowering-inducing gene in a site-specific manner usingsuch promoter, it is possible to induce the expression of the gene in aplant organ or plant tissue formed with cells having theflowering-inducing gene introduced thereinto so as to cause slowflowering induction.

The expression vector may further include a nucleic acid having adifferent segment sequence, in addition to a promoter and theflowering-inducing gene. The nucleic acid having a different segmentsequence is not particularly limited. Examples thereof include a nucleicacid having a terminator nucleotide sequence, a nucleic acid having atransformant selection marker nucleotide sequence, a nucleic acid havingan enhancer nucleotide sequence, and a nucleic acid having a nucleotidesequence for improving translation efficiency. In addition, therecombinant expression vector may further have a T-DNA region. A T-DNAregion can improve the efficiency of nucleic acid introductionespecially when introducing a nucleic acid having a nucleotide sequencein the recombinant expression vector into a plant cell usingAgrobacterium.

The nucleic acid having a terminator nucleotide sequence is notparticularly limited as long as it functions as a transcriptiontermination site, and it may be a known nucleic acid. For example,specific examples of such nucleic acid that can be used include thetranscription termination site of the nopaline synthase gene (Nosterminator) and the transcription termination site of cauliflower mosaicvirus 35S (CaMV35S terminator). Of these, the Nos terminator is used insome embodiments. The above-described recombinant vector can beprevented from causing an event of, for example, synthesizing anunnecessarily long transcript after being introduced into a plant cellby placing the terminator at an appropriate site.

Examples of the nucleic acid having a transformant selection markernucleotide sequence that can be used include nucleic acids havingdrug-resistant genes. Specific examples of such drug-resistant genesinclude drug resistant genes against, for example, hygromycin,bleomycin, kanamycin, gentamicin, and chloramphenicol. Accordingly, byselecting a plant that grows in a medium containing such antibiotics, atransformed plant can be readily selected.

Examples of the nucleic acid having a nucleotide sequence for improvingtranslation efficiency include a nucleic acid having the tobacco mosaicvirus-derived omega sequence. The expression efficiency of theabove-described flowering-inducing gene can be increased by placing thisomega sequence-containing nucleic acid in the untranslated region(5′UTR) upstream of the protein coding region. Thus, the above-describedrecombinant expression vector can contain nucleic acids having variousDNA segment sequences depending on the purpose.

The method for constructing a recombinant expression vector is also notparticularly limited. A nucleic acid having the above-described promoternucleotide sequence, the above-described flowering-inducing gene, and ifneeded, a nucleic acid having the above-described other DNA segmentsequences can be inserted into an appropriately selected vector to serveas a base in a certain order. For example, the flowering-inducing geneand a nucleic acid having a promoter nucleotide sequence (and if needed,for example, a nucleic acid having a terminator nucleotide sequence) areligated, thereby inserting the ligation product into the vector.

In addition, a method for propagating (producing) the expression vectoris also not particularly limited, and a conventionally known method canbe used. Usually, the vector can be propagated in Escherichia coli usedas a host. At such time, the type of Escherichia coli may be selecteddepending on the vector type in some embodiments.

Transformation

The expression vector is introduced into a plant cell of interest by anordinary transformation method. The method for introducing theexpression vector into a plant cell (transformation method) is notparticularly limited, and an appropriate conventionally known method canbe used depending on the plant cell. Specifically, for example, a methodusing Agrobacterium or a method in which the expression vector isdirectly introduced into a plant cell can be used. The method usingAgrobacterium that can be used is, for example, the method described inBechtold, E., Ellis, J. and Pelletier, G. (1993) In PlantaAgrobacterium-mediated gene transfer by infiltration of adultArabidopsis plants. C. R. Acad. Sci. Paris Sci. Vie, 316, 1194-1199 orthe method described in Zyprian E, Kado Cl, Agrobacterium-mediated planttransformation by novel mini-T vectors in conjunction with a high-copyvir region helper plasmid. Plant Molecular Biology, 1990, 15(2),245-256.

Examples of the method in which the expression vector is directlyintroduced into a plant cell that can be used include a microinjectionmethod, an electroporation method (electroporation method), apolyethylene glycol method, a particle gun method, a protoplast fusionmethod, and a calcium phosphate method.

In addition, when the method in which the flowering-inducing gene isdirectly introduced into a plant cell is employed, a nucleic acid havinga transcription unit such as a promoter nucleotide sequence or a nucleicacid having a transcription terminator nucleotide sequence, which isrequired for the expression of a flowering-inducing gene of interest,and a flowering-inducing gene of interest are necessary and sufficient,and the vector function is not necessary. Further, even a nucleic acidconsisting of the protein coding region of the flowering-inducing genewithout having a transcription unit is acceptable as long as it can beintegrated into a transcription unit of the host genome, therebyexpressing the gene of interest. Even in a case in which the nucleicacid cannot be integrated into the host genome, it is acceptable as longas the flowering-inducing gene is transcribed and/or translated in thecell.

Examples of plant cells into which the expression vector or aflowering-inducing gene of interest without the expression vector isintroduced include cells, callus, and suspension culture cells oftissues in plant organs such as flowers, leaves, and roots. Theexpression vector described herein may be appropriately constructed as avector suitable for the type of a plant to be produced or it may beconstructed as a versatile expression vector and then introduced into aplant cell.

A plant formed with cells into which an expression vector is introducedis not particularly limited. In other words, it is possible to causeslow flowering induction in any plant by introducing the above-describedflowering-inducing gene. A target plant is, for example, a floweringplant in some embodiments, and an angiosperm among flowering plants insome other embodiments. Examples of target angiosperms includedicotyledonous plants and monocotyledonous plants such as plantsbelonging to the families Brassicaceae, Poaceae, Solanaceae,Leguminosae, and Salicaceae (see below), but are not limited to theseplants.

Family Brassicaceae: thale-cress (Arabidopsis thaliana), lyraterockcress (Arabidopsis lyrata), rapes (Brassica rapa, Brassica napus,Brassica campestris), cabbage (Brassica oleracea var. capitata), napa(Brassica rapa var. pekinensis), qing-geng-cai (Brassica rapa var.chinensis), turnip (Brassica rapa var. rapa), Nozawana (Brassica rapavar. hakabura), Mizuna (Brassica rapa var. laciniifolia), Komatsuna(Brassica rapa var. perviridis), pak choy leaves (Brassica rapa var.chinensis), radish (Raphanus sativus), wasabi or Japanese-horseradish(Wasabia japonica), pink shepherd's-purse (Capsella rubella), and thelike

Family Chenopodiaceae: beet (Beta vulgaris)

Family Aceraceae: sugar maple (Acer saccharum)

Family Euphorbiaceae: castor bean (Ricinus communis)

Family Solanaceae: tobacco (Nicotiana tabacum), eggplant (Solanummelongena), potato (Solanum tuberosum), tomato (Solanum lycopersicum),pepper (Capsicum annuum), petunia (Petunia hybrida), and the like

Family Leguminosae: soybean (Glycine max), garden pea (Pisum sativum),broad bean (Vicia faba), Japanese wisteria (Wisteria floribunda), peanut(Arachis hypogaea), birdsfoot trefoil (Lotus japonicus), kidney bean(Phaseolus vulgaris), adzuki bean or English red mung bean (Vignaangularis), acacia (Acacia), barrelclover (Medicago truncatula),chickpea (Cicer arietinum), and the likeFamily Asteraceae: chrysanthemum (Chrysanthemum morifolium), sunflower(Helianthus annuus), and the likeFamily Arecaceae: oil palm (Elaeis guineensis, Elaeis oleifera), coconutpalm (Cocos nucifera), date palm (Phoenix dactylifera), wax palm(Copernicia), and the likeFamily Anacardiaceae: wax tree (Rhus succedanea), cashew tree(Anacardium occidentale), lacquer tree (Toxicodendron vernicifluum),mango (Mangifera indica), pistachio (Pistacia vera), and the likeFamily Cucurbitaceae: squash (Cucurbita maxima, Cucurbita moschata,Cucurbita pepo), cucumber (Cucumis sativus), Japanese snake gourd(Trichosanthes cucumeroides), bottle gourd (Lagenaria siceraria var.gourda), and the likeFamily Rosaceae: almond (Amygdalus communis), rose (Rosa), strawberry(Fragaria vesca), Japanese cherry (Prunus), apple (Malus pumila var.domestica), peach (Prunus persica), and the likeFamily Vitaceae: grape (Vitis vinifera)Family Caryophyllaceae: carnation (Dianthus caryophyllus) and the likeFamily Salicaceae: poplar (Populus trichocarpa, Populus nigra, Populustremula) and the likeFamily Poaceae: corn (Zea mays), rice (Oryza sativa), barley (Hordeumvulgare), bread wheat (Triticum aestivum), wild einkorn wheat (Triticumurartu), Tausch's goatgrass (Aegilops tauschii), Purple false brome(Brachypodium distachyon), Asian bamboo (Phyllostachys), sugarcane(Saccharum officinarum), Napier grass (Pennisetum purpureum), Erianthus(Erianthus ravennae), Japanese silver grass (Miscanthus virgatum),sorghum (Sorghum bicolor), switch grass (Panicum), and the likeFamily Liliaceae: tulip (Tulipa), lily (Lilium), and the like

Of these, plants belonging to the family Poaceae such as sugarcane,corn, Erianthus, rice, sorghum, and bread wheat, and in particular,plants belonging to the genus Saccharum, Erianthus, Sorghum, orMiscanthus are exemplified in some embodiments.

Other Steps and Methods

After the above-described transformation treatment, a selection step forselecting an appropriate transformant from among plants can be performedby a conventionally known method. The selection method is notparticularly limited. For example, selection may be carried out based ondrug resistance such as hygromycin resistance. Alternatively, aftergrowing transformants, the flowering induction time of each plant isobserved, and then, a plant in which flowering induction occurs earlierthan the wild-type plant but later than a transformed plant into which aconventionally known flowering-inducing gene has been introduced may beselected.

In addition, a progeny plant can be produced from a transformed plantobtained by the transformation treatment in accordance with an ordinarymethod. By selecting a progeny plant maintaining the characteristic thatthe expression level of the flowering-inducing gene is significantlyimproved as compared to the wild-type plant based on the floweringinduction time, it is possible to produce a plant line which stablymaintains the feature of inducing flowering slowly because of theabove-described characteristic. It is also possible to obtain aplant-propagating material such as plant cells, seeds, fruits, stock,callus, tuber, cut ears, or mass from a transformed plant or a progenythereof and produce a stable plant line having the characteristic usingsuch material on a large scale.

As described above, according to the present disclosure, it is possibleto cause slow flowering induction to occur by introducing theabove-described specific flowering-inducing gene into cells or enhancingthe expression of the flowering-inducing gene. In other words, floweringinduction can occur in a transformed plant into which theflowering-inducing gene has been introduced or a progeny thereof earlierthan the wild-type plant while the flowering induction occurs at a stagewhen the plant has grown more largely than a transformed plant intowhich a conventionally known flowering-inducing gene has beenintroduced. Accordingly, the amount of seeds of the transformed plantinto which the flowering-inducing gene has been introduced or a progenythereof does not decrease due to flowering induction at a stage when theplant is still small.

EXAMPLES

The present disclosure will be described in detail with reference to theExamples below. However, the scope of the present disclosure is notlimited to the Examples.

[FT Family Genes in Sugarcane]

In the Examples, a gene (flowering-inducing gene) encoding a florigendefined as a signal substance that induces flower bud formation insugarcane plants were searched for. FT (FLOWERING LOCUS T) family genesthat function as florigen genes have been identified for Arabidopsisthaliana and rice (see references listed at the end of the Examples:Kobayashi et al., 1999; Kardailsky et al., 1999; Corbesier et al., 2007;Tamaki et al., 2007; Komiya et al., 2007). However, no FT gene thatpromotes flowering induction has been identified in sugarcane (Coelho,2013; Coelho et al., 2014). Therefore, in the Examples, in order toidentify florigen genes (flowering-inducing genes) involved in floweringinduction in sugarcane, sugarcane FT family genes were searched for inthe existing databanks.

In the Examples, NCBI databases (EST, unigene, SRA), SUGARCANEFUNCTIONAL GENOMICS DATABASE(SUCEST-fun), Phytozome, plantGDB, MSU RiceGenome Annotation Project, and the Rice Annotation Project (RAP) wereused as databases for obtaining DNA sequence information and amino acidsequence information. In addition, the information on amino acidsequences encoded by candidate FT genes was obtained from a previouslypublished paper on sugarcane (Coelho et al., 2014). In the Examples, DNAand amino acid sequences obtained by performing homology search usingthe homology search program NCBI BLAST were subjected to lineageanalysis using EMBL-EBI Clustal Omega.

Specifically, three amino acid sequences ScFT3 to ScFT5 were obtainedfrom previously published papers disclosing FT genes in sugarcane(Coelho et al., 2013, Coelho et al., 2014). DNA fragment sequencescorresponding to these amino acid sequences were obtained from the NCBIdatabases and SUCEST. In addition to the above, BLAST search wasperformed using, as queries, information on the sequences of the Hd3agene (Kojima et al., 2002) and the ZCN8 gene (Meng et al., 2011) knownas florigen genes of rice and corn, respectively. As a result, inaddition to the information on partial DNA sequences of ScFT3 to ScFT5which were reported with their amino acid sequences in previouslyconducted studies, partial sequences of five kinds of FT family genes,which are a FT family gene similar to corn ZCN25 and ZCN19 (hereinafter,“ScFT6”), a FT family gene similar to ZCN18 and ZCN24 (hereinafter,“ScFT7”), a FT family gene similar to ZCN8 (hereinafter, “ScZCN8”), a FTfamily gene similar to ZCN12 (hereinafter, “ScZCN12”), and a FT familygene similar to ZCN16 (hereinafter, “ScZCN16”), were found.

[Primer Designing]

Primers for gene isolation were designed based on the partial sequenceinformation on the above-described FT family genes using Primer3(software provided by Whitehead Institute for Biomedical Research).

[Plant Material and Cultivation Method]

For cloning of the FT family genes, RNA obtained from the wild-typesugarcane (scientific name: Saccharum spontaneum L.; cultivar name: SES186; the Genebank Project, NARO) was isolated. Leaves, roots, apicalbuds, and stems of the wild-type sugarcane SES 186 were sampled beforeand after heading, followed by RNA extraction. Before heading, samplesof leaves were collected from each individual which had been cultivatedfor 17 months under long-day conditions supplemented with natural light(light period of 16 hours, dark period of 8 hours, about 28° C.). Eachleaf was taken from the top portion at a level of 30 cm from the tip ofthe third leaf such that a leaf sample having a length of about 1.5 cmwas obtained from one side of the leaf while excluding the costa. Afterheading, samples of leaves were obtained from both individuals at theearly stage of heading when a 1-cm ear came out from the root of a flagleaf of each plant cultivated under short-day conditions without lightsupplementation and individuals cultivated for 4 weeks after heading.Samples of roots, apical buds, and stems were collected from seedlingplants cultivated under long-day conditions (light period of 16 hours,dark period of 8 hours, about 28° C.) after redifferentiation of callusinduced from the growth point. A root sample used herein was the whiteportion of a root including the tip, an apical bud sample used hereinwas the portion of an apical bud including the uppermost node with avery little portion of a leaf sheath extending from the node, and a stemsample used herein was the portion including the second and third nodescounted from the lowest node of a stem.

[RNA Extraction]

Total RNA was purified from sugarcane samples (leaf: 15-30 mg; root:20-30 mg; apical bud: about 35 mg; stem: 15-55 mg) using a RNeasy PlantMini kit (QIAGEN) basically in accordance with the method recommended bythe manufacturer. Incorporated genome DNA was removed using a RNase-freeDNase set (QIAGEN) basically in accordance with the method recommendedby the manufacturer. Thereafter, the amount of RNA was quantitativelydetermined by NanoDrop ND-1000 (Thermo Fisher Scientific), therebydetermining the final concentration.

[Gene Isolation]

cDNA was synthesized using the extracted RNA. cDNA was synthesizedbasically in accordance with the method recommended by the manufacturerusing a PrimeScript RT reagent kit (Takara Bio Inc.) and the extractedRNA (500 ng) as a template. Thereafter, PCR was performed as describedbelow so as to analyze the nucleotide sequence of each amplified DNAfragment.

PCR was performed using PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) asfollows: 38 cycles of denaturation at 98° C. for 10 seconds, annealingat 55° C. for 10 seconds, and extension reaction at 72° C. for 1 minute.Further, PCR was performed using ExTaq HS (Takara Bio Inc.) as follows:40 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C.for 30 seconds, and extension reaction at 72° C. for 1 minute. PCRproducts were subjected to agarose gel electrophoresis and the resultingbands were excised and purified. A pENTR/D-TOPO vector (Thermo FisherScientific) or poly(A) was added to each fragment amplified by PrimSTARHS, and then the fragment was subcloned into a pGEM-T Easy vector(Promega), followed by DNA sequence analysis. Each experimentalcondition followed the instructions of the kits and reagents. For eachFT family gene for which full-length sequence information was notobtained by PCR, the full-length sequence was isolated by the Racemethod and identified. Cloning of the 5′ end was performed using aSMARTer RACE 5′/3′Kit (Takara Bio Inc.). Cloning of the 3′ end wasperformed using a 3′-Full RACE Core Set (Takara Bio Inc.).

TABLE 1 Primer Amplified SEQ ID No. Name of Primer gene Primer SequenceNO: 1 attB1_AT1G65480_F AtFT ggggacaagtttgtacaaaaaagcaggcttcATGT 5CTATAAATATAAGAGACCCTCTTAT 2 attB2_AT1G65480_R AtFTggggaccactttgtacaagaaagctgggttAAGTC 6 TTCTTCCTCCGCAGCCACTCTCCCT 3ScFT-like-F2 ScFT3 YTIMGIGARTAYYTICAYTGGYTIGT 7 ScZCN8 4 ScFT-like-R1ScFT3 TRAARTTYTGICKCCAICCIGGIGC 8 ScZCN8 5 ScFT3-TO-F149 ScFT3CACCCGTCGGTGGCCCATTATTG 9 6 ScFT3-TO-R757 ScFT3 TCTTATTTCACCCGGATCGAGT10 7 attB1_ScZCN8_F ScZCN8 ggggacaagtttgtacaaaaaagcaggctccATGT 11CAGCAACCGATCCTTTGGTCATGGC 8 attB2_ScZCN8_R ScZCN8ggggaccactttgtacaagaaagctgggtcCTACT 12 CTTCCCTAAACCTTCTTCCACCCGA 9attB1_ScFT4_F1 ScFT4 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGG 13CCGGCAGCGGCAGGGAAAGGGAGAC 10 attB2_ScFT4_R4 ScFT4GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATG 14 AGTACATCCTCCTTCCCCCGGAGCC 11attB1_ScFT5 ScFT5 ggggacaagtttgtacaaaaaagcaggctccATGT 15TCAATATGTCTAGGGACCCATTGGT 12 attB2_ScFT5 ScFT5ggggaccactttgtacaagaaagctgggtcTCACC 16 TTATGTACCTTCTTCCACCACAGCC 13attB_ScZCN12_F1 ScZCN12 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGT 17CTCAGGTGGAACCGTTGGTTCTGGT 14 attB2_ScZCN12_R4 ScZCN12GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACG 18 AACTTTCGGGCCTGAACCTTCTGCC 15attB1_ScZCN16 ScZCN16 ggggacaagtttgtacaaaaaagcaggctccATGT 19CAAGTGACCCACTTGTTGTAAGCAA 16 attB2_ScZCN16 ScZCN16ggggaccactttgtacaagaaagctgggtcTCATC 20 TAATATATCGTCTGCCACCGCACCC 17ScFT6-TO-F13 ScFT6 CACCCAAAATTAGCAGTCTTGACTAACC 21 18 ScFT6-TO-R582ScFT6 GCACAGTCAGTGAGATGGTAT 22 19 ScFT7-TO-Met ScFT7CACCATGTCACGAGGCAGGGATCC 23 20 ScFT7_TO_R338 ScFT7 TACTGCCTTGACGTCGATGT24 21 HPTII_semiRT_F HPTII CGACGTCTGTCGAGAAGTTTC 25 22 HPTII_semiRT_RHPTII ATTCCTTGCGGTCCGAATGG 26

[Preparation of Vector for Arabidopsis thaliana Transformation]

Next, a vector used for introducing the above-described FT family genesinto Arabidopsis thaliana was prepared. Specifically, at first, DNAfragments each containing a full-length coding sequence were obtained bythe PCR method described below based on the DNA sequence informationobtained by the method described above for the above-described FT familygenes. Next, the obtained DNA fragments were separately incorporatedinto pDONR207 (Thermo Fisher Scientific) or pENTR/D-TOPO (Thermo FisherScientific), thereby producing entry clones. Subsequently, each DNAfragment including one coding region was incorporated into a binaryvector pDEST_35S_HSP_GWB5 by LR reaction using the entry clone and LRclonase II (Thermo Fisher Scientific), thereby producing a vector forArabidopsis thaliana transformation. The vector for Arabidopsis thalianatransformation strongly expresses the corresponding FT family geneconstantly under control of the CaMV 35S promoter and the HSP terminatorin Arabidopsis thaliana.

pDEST_35S_HSP_GWB5 was prepared by cleaving a fragment containingattR4-ccdB-attR2-SRDX of R4pGWB5_SRDX_HSP (Oshima et al., 2011) byrestriction enzyme HindIII treatment and inserting a fragment containing35S-Ω-attR1-ccdB-attR2 obtained by treating pDEST35SHSP (Oshima et al.,2013) with HindIII into the cleavage site.

Hereinafter, detailed procedures for preparing an entry clone for eachgene will be described.

<Atft Gene>

In the Examples, a transformant that overexpresses the FT gene (AtFTgene) from Arabidopsis thaliana was prepared for comparison. Prior topreparing a vector for Arabidopsis thaliana transformation forintroducing the AtFT gene, primers (Primer Nos. 1 and 2) havingsequences for adding the attB1 and attB2 sequences were designed basedon the nucleotide sequence of the AtFT gene disclosed with NCBIAccession No. NM_105222. An amplified fragment (SEQ ID NO: 27) includingthe AtFT gene was obtained by PCR using these primers. The obtainedamplified fragment was incorporated into pDONR207 (Thermo FisherScientific) by BP reaction using BP clonase II (Thermo FisherScientific), thereby preparing an entry clone for the AtFT gene.

<Oshd3a Gene>

In the Examples, a transformant that overexpresses the FT gene (OsHd3agene) from rice was prepared for comparison. Prior to preparing a vectorfor Arabidopsis thaliana transformation for introducing the OsHd3a gene,VectorBuilder Inc. was commissioned to carry out artificial synthesis ofa nucleic acid fragment (SEQ ID NO: 28) to which the attB1 sequence wasadded to the 5′ end side of the coding sequence and the attB2 sequencewas added to the 3′ end side of the same based on the nucleotidesequence of the OsHd3a gene disclosed with NCBI Accession No. AB052944and incorporation of the fragment into RI201-AN (Takara Bio Inc.). Anentry clone was prepared for the OsHd3a gene by BP reaction using theobtained plasmid and pDONR207 (Thermo Fisher Scientific) using BPclonase II (Thermo Fisher Scientific).

<ScFT3 and ScZCN8 Genes>

For the ScFT3 gene and the ScZCN8 gene among the sugarcane-derived FTfamily genes isolated as described above, a partial fragment wasobtained by degenerate PCR using degenerate primers (Primer Nos. 3 and4), and the full-length sequence was further obtained by degenerate PCRand the Race method. In addition, the ScFT3 gene was cloned intopENTR/D-TOPO (Thermo Fisher Scientific) and the ScZCN8 gene was clonedinto pDONR207 (Thermo Fisher Scientific), thereby preparing an entryclone for each of the ScFT3 gene and the ScZCN8 gene.

For the ScFT3 gene, the full-length sequence necessary for entry clonepreparation was amplified using Primer Nos. 5 and 6. For the ScZCN8gene, the full-length sequence necessary for entry clone preparation wasamplified using Primer Nos. 7 and 8. The nucleotide sequences ofamplified DNA fragments were as follows: ScFT3 gene: SEQ ID NO: 29; andScZCN8 gene: SEQ ID NO: 30.

<ScFT4, ScFT5, ScFT6, ScFT7, ScZCN12, and ScZCN16 Genes>

For the ScFT4, ScFT5, ScFT6, ScFT7, ScZCN12, and ScZCN16 genes among thesugarcane-derived FT family genes isolated as described above, cloningwas performed based on the nucleotide sequence information of thedatabases, thereby obtaining full-length sequences.

For each of the ScFT4, ScFT5, ScZCN12, and ScZCN16 genes, primers foradding the attB1 sequence to the 5′ side of the sequence and the attB2sequence to the 3′ end of the same were designed. Specifically, thefollowing primers were designed: ScFT4 gene: Primer Nos. 9 and 10; ScFT5gene: Primer Nos. 11 and 12; ScZCN12 gene: Primer Nos. 13 and 14; andScZCN16 gene: Primer Nos. 15 and 16. The full-length sequences necessaryfor preparing entry clones each including one of the genes wereamplified by PCR using these primers. The nucleotide sequences ofamplified DNA fragments were as follows: ScFT4 gene: SEQ ID NO: 31;ScFT5 gene: SEQ ID NO: 32; ScZCN12 gene: SEQ ID NO: 33; and ScZCN16gene: SEQ ID NO: 34. Each obtained DNA fragment was incorporated intopDONR207 (Thermo Fisher Scientific) by BP reaction, thereby preparing anentry clone for each gene.

Meanwhile, for the ScFT6 gene and the ScFT7 gene, primers in which caccwas added to the 5′ side were designed. Specifically, the followingprimers were designed: ScFT6 gene: Primer Nos. 17 and 18; and ScFT7gene: Primer Nos. 19 and 20. The full-length sequences necessary forpreparing entry clones each including one of the genes were amplified byPCR using these primers. The nucleotide sequences of amplified DNAfragments were as follows: ScFT6 gene: SEQ ID NO: 35; and ScFT7 gene:SEQ ID NO: 36. Each obtained DNA fragment was incorporated intopENTR/D-TOPO (Thermo Fisher Scientific), thereby preparing an entryclone for each of the ScFT6 gene and the ScFT7 gene.

[Functional Evaluation of FT Family Genes in Arabidopsis thaliana]

For functional evaluation of the isolated sugarcane-derived FT familygenes, transformation of Arabidopsis thaliana (Arabidopsis) was carriedout using the above-described binary vector, thereby analyzing floweringcharacteristics.

[Transformation Using Agrobacterium]

The above-described binary vector plasmid was transformed intoAgrobacterium (Agrobacterium tumefaciens, Rhizobium radiobacter) GV3101by the electroporation method and cultured in an LB medium containing 50mg/l spectinomycin, 50 mg/l gentamicin, and 50 mg/l rifampicin.Accordingly, Agrobacterium transformed with the binary vector wasprepared.

[Preparation of Arabidopsis thaliana Transformants]

The Arabidopsis thaliana FT function-deficient strain ft-10 (Yoo et al.2005, Kleinboelting et al. 2012) was procured from the ArabidopsisBiological Resource Center (ABRC). Basically, the wild-type Col-0 strainand the ft-10 strain of Arabidopsis thaliana, each strain forming buds,were transformed by the floral dipping method described in Clough & Bent(1998). In addition, transformation was carried out using Agrobacteriumincluding the binary vector pDEST_35S_HSP_GWB5 that had not beensubjected to LR reaction, thereby preparing a control. In order toselect transformed plants, T1 seeds obtained from the plants treated bythe floral dipping method were sterilized and seeded in an MS mediumcontaining 30 mg/l hygromycin and 250 mg/l vancomycin (Murashige andSkoog, 1962, including 0.5% sucrose and 0.8% agar). The medium afterseeding was left at 4° C. for 3 days for low-temperature treatment,followed by cultivation at 22° C. under long-day conditions (lightperiod of 16 hours/dark period of 8 hours). On day 15 after the start ofcultivation, individuals that survived antibiotics selection weretransplanted as transformed plants onto culture soil. Cultivation wascontinued after transplantation at 22° C. under long-day conditions(light period of 16 hours/dark period of 8 hours).

[Investigation of Flowering Time]

For the investigation of flowering time of T1 plants, transformedArabidopsis thaliana individuals were cultivated at 22° C. underlong-day conditions (light period of 16 hours/dark period of 8 hours),and the number of days until flower bud formation was observed and thenumber of leaves formed by that time (stem leaves and rosette leaves)were counted.

In addition, T2 seeds were collected from the T1 plants cultivated at22° C. under long-day conditions, and the flowering time under short-dayconditions was investigated. It is known that Arabidopsis thaliana isless likely to bloom under short-day conditions (light period of 8hours/dark period of 16 hours, 22° C.). It was therefore expected toeasily judge the results of flowering regulation by comparing theresults with the results obtained under long-day conditions.Specifically, for the investigation of flowering time of T2 plants undershort-day conditions, T2 seeds were allowed to absorb water andlow-temperature treated at 4° C. for 3 days and then seeded directly onculture soil. At this time, transformed plants were selected by genomicPCR described below.

[Genomic PCR]

Genome DNA was extracted by a simplified DNA extraction method fromrosette leaf pieces of Arabidopsis thaliana sampled from early bloomingindividuals after flowering and the other individuals 30 to 60 daysafter seeding directly on culture soil. DNA was extracted by disruptingthe leaf pieces in extraction buffer (200 mM Tris-HCl (pH 7.5), 250 mMNaCl, 25 mM EDTA), and purifying the centrifuged supernatant byisopropanol precipitation, concentrating the resultant, and suspendingthe concentrate in TE buffer. The extracted DNA was used as template forPCR. Primers (Primer Nos. 21 and 22) specific to thehygromycin-resistant gene HPTII present on the T-DNA sequence of thebinary vector used for transformation and TaKaRa ExTaq (Takara Bio Inc.)were used for carrying out PCR by the method recommended by themanufacturer, and individuals from which amplified fragments wereobtained were determined to be transformed plants.

[Measurement of the Amount of Seeds]

The amount of seeds was measured by weighing seeds collected from eachof transformed plants dried for about a month after stopping irrigationusing a precision balance (Excellence Plus, Metller Toledo).

[Results]

FIG. 1 depicts the results of investigating flowering time bycultivating transformed plants, in which the FT family genes wereoverexpressed, using the Arabidopsis thaliana FT function-deficientstrain ft-10 as a host under long-day conditions. As depicted in FIG. 1,for the vector control strain of the FT function-deficient strain ft-10,about 50 true leaves were formed before flower bud formation wasobserved, and the characteristic that flower bud formation is delayedfor a long period of time due to FT function deficiency was confirmed.Meanwhile, flower buds were formed in transformed plants in which theconventionally known AtFT gene or OsHd3a gene was overexpressed whenabout 5 true leaves were formed, indicating that the FT function wascomplemented by the introduced gene.

For the ScFT3, ScFT4, ScFT5, ScFT7, ScZCN8, and ScZCN12 genes among theFT family genes isolated in the Examples, it was revealed that the genesfunction to complement the FT function to induce flower bud formation ata very early stage as with the AtFT gene and the OsHd3a gene as depictedin FIG. 1. Meanwhile, for the ScFT6 and ScZCN16 genes among the FTfamily genes isolated in the Examples, it was revealed that flower budformation is induced earlier than that in the vector control strain ofthe FT function-deficient strain ft-10, while flower bud formation isinduced relatively slowly, which is remarkably different from the AtFTgene, the OsHd3a gene, and the other FT family genes isolated in theExamples.

FIG. 2 depicts the results of measuring the weight of seeds perindividual transformed plant for investigating the seed yields oftransformed plants cultivated in the same manner. As depicted in FIG. 2,it was found that in the case of introducing the ScFT6 or ScZCN16 geneamong the FT family genes isolated in the Examples, the seed yield isremarkably improved compared with the case of introducing any of theAtFT gene, the OsHd3a gene, and the other FT family genes isolated inthe Examples. In particular, it was found that in the case ofintroducing the ScZCN16 gene, the seed yield reaches the level of thevector control strain, and in the case of introducing the ScFT6 gene,the seed yield reaches a level significantly higher than the level ofthe vector control strain.

As described above, it was shown that in the case of introducing theScFT6 or ScZCN16 gene among the FT family genes isolated in theExamples, flower bud formation can be induced earlier than that in thevector control strain, and the improved seed yield can be achieved.

FIGS. 3 and 4 depict the results of investigating flowering time bycultivating transformed plants, in which the FT family genes wereoverexpressed, using the wild-type Arabidopsis thaliana as a host undershort-day conditions. The results for two T2 lines (#1 and #2 in thefigure) obtained from two different T1 individuals are shown except forthe vector control. As depicted in FIGS. 3 and 4, for the vector controlstrain of the wild-type Arabidopsis thaliana, about 60 true leaves wereformed before flower bud formation was observed, and the characteristicthat flower bud formation is delayed under short-day conditions wasconfirmed under short-day conditions that are less likely to causeblooming than long-day conditions. Meanwhile, flower buds were formed intransformed plants in which the conventionally known AtFT gene or any FTfamily gene other than the ScFT6 and ScZCN16 genes was overexpressedwhen about 5 to 10 true leaves were formed, indicating that flower budformation is obviously induced by the introduced gene.

On the other hand, it was revealed that flower bud formation is inducedearlier in the transformed plants obtained by introducing the ScFT6 orScZCN16 gene into wild-type Arabidopsis thaliana than that in the vectorcontrol strain (i.e., the wild-type strain), while flower bud formationis induced relatively slowly, which is remarkably different fromtransformed plants obtained by introducing the AtFT gene or any other FTfamily gene.

REFERENCES

Clough S J, Bent A F. (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.16(6):735-743.

-   Coelho C P (2013) MOLECULAR REGULATORY MECHANISM OF FLORAL    TRANSITION BY FT/TFL1 ORTHOLOGS AND THE AUTONOMOUSLY EXPRESSED ScID1    MONOCOT-SPECIFIC TRANSCRIPTION FACTOR IN SUGARCANE. Thesis.    Universidade Federal de Lavras-   Coelho C P, Minow M A, Chalfun-Junior A, Colasanti J. (2014)    Putative sugarcane FT/TFL1 genes delay flowering time and alter    reproductive architecture in Arabidopsis. Front Plant Sci. 5:221.-   Corbesier L1, Vincent C, Jang S, Fornara F, Fan Q, Searle I,    Giakountis A, Farrona S, Gissot L, Turnbull C, Coupland G. (2007) FT    protein movement contributes to long-distance signaling in floral    induction of Arabidopsis. Science. 316(5827):1030-1033.-   Kardailsky I, Shukla V K, Ahn J H, Dagenais N, Christensen S K,    Nguyen J T, Chory J, Harrison M J, Weigel D. (1999) Activation    tagging of the floral inducer FT. Science. 286(5446): 1962-1965.-   Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T. (1999) A pair of    related genes with antagonistic roles in mediating flowering    signals. Science. 286(5446):1960-1962.-   Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T,    Yano M. (2002) Hd3a, a rice ortholog of the Arabidopsis FT gene,    promotes transition to flowering downstream of Hd1 under short-day    conditions. Plant Cell Physiol. 43(10): 1096-1105.-   Komiya R, Ikegami A, Tamaki S, Yokoi S, Shimamoto K (2008) Hd3a and    RFT1 are essential for flowering in rice. Development.    135(4):767-774.-   Kleinboelting N, Huep G, Kloetgen A, Viehoever P, Weisshaar B (2012)    GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana    T-DNA mutant database. Nucleic Acids Res. 40:D1211-D1215.-   Meng X, Muszynski M G, Danilevskaya O N. (2011) The FT-like ZCN8    Gene Functions as a Floral Activator and Is Involved in Photoperiod    Sensitivity in Maize. Plant Cell. 23(3):942-960.-   Murashige T, Skoog F K (1962) A revised medium for rapid growth and    bio-assays with tobacco tissue cultures. Physiol. Plant 15 (3):    473-497.-   Oshima Y, Mitsuda N, Nakata M, Nakagawa T, Nagaya S, Kato K,    Ohme-Takagi M. Novel vector systems to accelerate functional    analysis of transcription factors using chimeric repressor    gene-silencing technology (CRES-T). (2011) Plant Biotech. 28:201-10.-   Oshima Y, Shikata M, Koyama T, Ohtsubo N, Mitsuda N, Ohme-Takagi M.    MIXTA-like transcription factors and WAX INDUCER1/SHINE1    coordinately regulate cuticle development in Arabidopsis and Torenia    fournieri. (2013) Plant Cell. 25:1609-24.-   Tamaki S, Matsuo S, Wong H L, Yokoi S, Shimamoto K. (2007) Hd3a    protein is a mobile flowering signal in rice. Science.    316(5827):1033-1036.-   Tsuruta S I, Ebina M, Kobayashi M, Takahashi W. (2017) Complete    Chloroplast Genomes of Erianthus arundinaceus and Miscanthus    sinensis: Comparative Genomics and Evolution of the Saccharum    Complex. PLoS One. 12(1):e0169992.-   Yoo S K, Chung K S, Kim J, Lee J H, Hong S M, Yoo S J, Yoo S Y, Lee    J S, Ahn J H. (2005) CONSTANS activates SUPPRESSOR OF OVEREXPRESSION    OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in    Arabidopsis. Plant Physiol. 139(2):770-778. Wu Z Q, Ge S (2012) The    phylogeny of the BEP clade in grasses revisited: Evidence from the    whole-genome sequences of chloroplasts. Mol Phylogenet Evol.    62(1):573-578.    SEQUENCE LISTING

What is claimed is:
 1. A transformed plant or transformed plant cell,wherein a flowering-inducing gene encoding the following protein of (a)or (b) has been introduced thereinto or which an expression of theflowering-inducing gene present as an endogenous gene has been enhanced:(a) a protein comprising the amino acid sequence of SEQ ID NO: 4; or (b)a protein comprising an amino acid sequence having 98% or more identityto the amino acid sequence of SEQ ID NO: 4 and having ability to induceflowering.
 2. The transformed plant or transformed plant cell accordingto claim 1, which belongs to the family Poaceae.
 3. The transformedplant or transformed plant cell according to claim 1, which belongs tothe genus Saccharum, Erianthus, Sorghum, or Miscanthus.
 4. A floweringinduction method, comprising introducing an expression vector comprisinga flowering-inducing gene encoding the following protein of (a) or (b):(a) a protein comprising the amino acid sequence of SEQ ID NO: 4; or (b)a protein comprising an amino acid sequence having 98% or more identityto the amino acid sequence of SEQ ID NO: 4 and having ability to induceflowering.
 5. The flowering induction method according to claim 4, whichcomprises introducing the flowering-inducing gene into a plant belongingto the family Poaceae.
 6. The flowering induction method according toclaim 4, which comprises introducing the flowering-inducing gene into aplant belonging to the genus Saccharum, Erianthus, Sorghum, orMiscanthus.